Improving our understanding of the early immunological responses to influenza A virus (IAV) is significant due to the annual IAV morbidity and mortality, the risk of a catastrophic new pandemic and the limitations of current vaccines and therapeutics. Many aspects of the development of protective immunity and the maintenance of immune homeostasis in the human lung are largely unknown. Our central theme is that the early immune response to the initial IAV infection in human lung is an emergent property from many cell types, host processes, pathogen effects, and microenvironment factors, propagating across scales and interacting in space and time. How stochastic processes and the resulting single cell response variation influence tissue level immunological response is an important question we are poised to address. Predictive immunological modeling, which is needed to understand this complex system, requires a collaborative program to anchor models in detailed human time course immunological data obtained in primary human cells and human lung tissue. Our existing NIAID contract-funded program (PRiME) for modeling immunity for biodefense has studied the dynamic immunological responses of human monocyte-derived dendritic cells (mo-DC) to IAV, providing new insights into host, virus and stochastic mechanisms that operate in mo-DCs following infection. We now propose to advance predictive modeling of IAV infection in humans by significantly expanding our focus to include multiple cell types that interact in space and time--experimentally-validated models will be developed for the human tracheobronchial epithelial cells (HTBE), which are the initial line of defense against virus, and the primary CD1c+ human DC subtype cells which respond to IAV during the first days of infection to contribute to the immediate immune response and to initiate adaptive immunity. Model parameterization and validation are enabled by new key technologies we have established, including barcoded recombinant viruses, single cell assays, human lung explants and multiscale modeling methods. Project I will quantify the responses of fully differentiated primary HTBE to IAV infection. Projec 2 will quantify the responses of primary CD1c+ DC and fresh human lung tissue to IAV infection. Project 3 will develop multiscale models of IAV infection in HTBE and DC in culture and in the context of the lung microenvironment. The wild-type and recombinant viruses studied will be generated in Core B: Virology. The immune assays will be standardized for all experimental projects by Core C: Immune Assay. The data analysis, data handling and dissemination of models and data will be facilitated by Core D: Model and Data Management. Central to all experimental and modeling projects and service cores is Core A: Administrative, which will coordinate all program activities and develop educational programs. This research program will improve the understanding of the mechanisms underlying the immune response to IAV in order to provide the basis for improved strategies for therapeutics and vaccination.